The cells of higher organisms are subjected to a continuing exposure to a variety of xenobiotic hostile chemical agents. Many of these chemical agents are pro-oxidants, and free oxygen radicals are associated with detrimental events in many cell types. Accordingly, living organisms have naturally evolved biochemical detoxification mechanisms to cope with substances to which they are exposed in their environment.
Thus, higher eukaryotic life forms possess several gene families whose main purpose is detoxification of noxious chemicals. Notable among these are the glutathione S-transferases (GST) which are a ubiquitous family of isoenzymes present in all known eukaryotic species in a variety of cytosolic and membrane bound isoenzymes. Each of the GST isoenzymes displays distinct catalytic and non-catalytic binding activities and are present in many isozymes in each individual. The study of GSTs has evolved to such an extent that the cytosolic enzymes are divided into at least five distantly related gene families. Individuals have multiple isozymes from many of the families in each of their cells. The expression of GST enzymes in a given cell is stimulated by a structurally diverse range of xenobiotic agents. At lest 100 chemicals have been found that induce GST activity. A significant number of these chemical gene inducers occur naturally and are found as non-nutritive components in vegetables and citrus fruits. It has become apparent, after much study, that several systems exist in parallel to induce the transcriptional activity of GST genes, including the xenobiotic-responsive element (XRE) and the glucocorticoid-responsive element (GRE). Another well studied gene induction system which induces GST activity is known as the antioxidant-responsive element (ARE). ARE is a system active at a transcriptional level for up regulating the expression of several categories of chemoprotective enzymes in response to induction by a chemical agent.
Separately, epidemiological studies of human cancer revealed that certain substances to which humans are exposed have a statistically deterrent effect on the occurrence of cancers in human populations. Inhibition of chemical carcinogenesis by pre-exposure to protective chemical compounds was first observed over 65 years ago. One mechanism by which such compounds actuate their protective effect is by the induction of expression of GSTs. A wide variety of protective compounds have been shown to induce increased GST activity in cells and tissues. Examples of such substances include sulforaphane, natively found in cruciform vegetables such as broccoli, Oltipraz, an antischistosomal drug, and butylhydroxyanisole, or BHA, a common food preservative. The statistical demonstration that ingestion of such substances was associated with a diminution of risk of cancer led researchers into the investigation of the molecular biology of interaction of the active agents from such foods and the cancer suppression process. Thus, for example, in Wattenberg "Chemoprevention of Cancer" Cancer Research 45:1-8 (1985), the effect of many chemopreventive agents is discussed.
It was later proposed, and then later demonstrated to be correct, that a transcriptional activator was responsible for the initiation of chemoprotective gene families in eukaryotic cells. Early suggestions focused on the fact that multiple forms of chemical inducers could result in the induction of cancer protective enzymes. Prochaska et al., Proc. Natl. Acad. Sci. USA, 82:8232-8236 (1985). Later a specific antioxidant responsive element (ARE) was identified in the 5' flanking region of the rat glutathione S transferase Ya subunit gene. Rushmore et al., J. Biol. Chem., 266:18:11632-11639 (1991). It was found that the ARE is responsive to a subset of antioxidants and also responsive to compounds linked to reactive oxygen species. The ARE was found to be part of a signal transduction pathway that allows eukaryotic cells to sense and respond to oxidation stress.
It was also determined that the pathway of chemoprotection is a multistep detoxification process involving at least two phases, an oxidation phase (phase I), a detoxification phase (phase II), and, possibly, a transport phase (phase III). The GST family of detoxification enzymes is induced by either of bifunctional inducers, which induce both phase I and phase II activities and by monofunctional inducers which regulate only phase II activity. Sulforaphone, Oltipraz and BHA are all monofunctional phase II inducers. Induction of the phase II enzymes GST Ya (GST1--1), NAD(P)H:quinone oxidoreductase (QR) and heme oxygenase-1 (HO-1) was shown to occur at the level of transcription. A regulatory element that mediates this increase in transcriptional rate was identified in the promoters of the rat and mouse GST Ya genes, the rat and human QR genes, and the mouse HO-1 gene. This chemoprotector inducible element has been termed the Antioxidant Responsive Element (ARE), or alternatively, the Electrophile Responsive Element. Rushmore et al. J. Biol. Chem., 265:14648-14653 (1990); Friling et al. Proc. Natl. Acad. Sci. USA, 87:3826-3830 (1900).
Further work refined the ARE to a 30-bp element in the QR promoter and to a 41-bp element in the GST Ya promoter. All of the described ARE's were found to contain a minimal core sequence defined to be RTGACnnnGC. Daniel, Crit. Rev. Biochem. Mol. Biol., 28:173-207 (1993). The term ARE core sequence, as used here, refers to this 10 bp sequence.